The human innate immune system has developed complex responses to viral infections to prevent the spread of pathogens. A newly discovered cellular membrane restriction factor, named tetherin, retains nascent HIV-1 virions at the cell surface. To combat tetherin, HIV-1 has evolved a mechanism that involves its accessory protein, Vpu, to trigger retroviral particle release. Structural analysis of tetherin and HIV-1 Vpu will provide significant insight into the functions of these proteins. However, tetherin and HIV-1 Vpu are integral membrane proteins that may pose challenges to crystallization and subsequent structural analysis. Small angle X-ray scattering (SAXS) is a rapidly developing technique that is ideally suited to provide low- resolution molecular 3D reconstructions of difficult macromolecular targets, such as integral membrane proteins and protein complexes and assemblies, without the need for crystals. This K99/R00 NIH Pathway to Independence award will allow me to first learn SAXS from a unique team of expert mentors in the laboratories of Dr. Erica Ollmann Saphire and Dr. John A. Tainer. SAXS will complement my current expertise in crystallography and allow me to determine hybrid pseudo-atomic resolution models of proteins involved in retroviral release and pathogenesis in my future independent faculty research. In the R00 Independent Phase, the following specific aims are designed to provide the critical preliminary results to initiate structural analysis of tetherin and HIV-1 Vpu: Specific Aim (1): Expression and crystallization of tetherin for structural analysis. Here, tetherin will be expressed in HEK293T cells, characterized and screened for crystallization. Specific Aim (2): Structural analysis of the oligomeric HIV-1 Vpu ion channel. Full-length HIV-1 Vpu will be expressed in HEK293T and E. coli cells, detergent solubilized from membranes and screened for crystallization. SAXS 3D reconstructions will be performed to provide an initial oligomeric Vpu model. I anticipate this K99/R00 award will allow me to successfully start an independent research program and to provide the preliminary data necessary for submission of a NIH R01 application by the end of the award. RELEVANCE (See instructions): While HIV-1 viral entry is well documented, the mechanism of viral release is poorly defined. No structures of tetherin and oligomeric HIV-1 Vpu are available, and any new structures of tetherin or HIV-1 Vpu will deliver astonishing insight into understanding the mechanisms of viral tethering, contribute to the discovery of important fundamental principles and is key to developing new classes of HIV-1/AIDS inhibitors.